The overall objective of the project is to study the dietary and hormonal regulation of fatty acid metabolism. Diabetes mellitus decreases fatty acid synthesis by decreasing the synthesis of fatty acid synthetase (FAS). We plan to study the role of insulin and related hormones on the synthesis of fatty acid synthesis of fatty acid synthetase in intact animals and in cultured cells. Specific antibodies against FAS are labeled with 125I and used for the isolation of polysomes associated with nascent polypeptide chains of FAS. The levels of polysomes bound to 125I-anti FAS are altered by diet and insulin in intact animals and in liver explants, and by insulin, glucagon and cAMP in developing chick embryo. Attempts will be made to isolate and characterize the mRNA of the synthetase. The large size of the FAS mRNA will be of value in developing procedures for its isolation. Translation of the mRNA will be followed using the reticulocyte lysate or the wheat germ systems. The effect of insulin on the stability of the mRNA and its translation by the polysomes will be studies. Liver explants and mammary glands grown in chemically defined medium permit us to study the direct effects of insulin on the synthesis of FAS and its mRNA. The effect of insulin, cortisol and prolactin on the induction of fatty acid synthetase and the lipogenic enzymes in mammary gland explants will be investigated. Specifically induced enzymes such as the thioesterase II and the triglyceride synthesizing enzymes of mammary gland will be isolated and their interaction with the FAS will be determined. Insulin induction of some of these enzymes, especially the thioesterase, will be studied in detail both at the messenger and enzyme levels. Methodology to be employed in the outlined studies include enzymology, protein chemistry, lipid chemistry and cell biology.